Packaging cells

ABSTRACT

The invention described herein allows the production of recombinant retroviruses (retroviral vector particles) from producer cells which are safer and of higher titer than normal. In addition, methods are provided for making helper cells which, when a recombinant retrovirus genome is introduced to make a producer line, produce particles that are targeted toward particular cell types. Methods are also provided for making recombinant retrovirus systems adapted to infect a particular cell type, such as a tumor, by binding the retrovirus or recombinant retrovirus in the particular cell type. Methods are also provided for producing recombinant retroviruses which integrate in a specific small number of places in the host genome, and for producing recombinant retroviruses from transgenic animals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/389,525, filed Sept. 2, 1999, now abandoned; which is a continuationof U.S. application Ser. No. 09/012,328, filed Jan. 23, 1995 nowabandoned; which is a divisional of U.S. application Ser. No.08/837,605, filed Apr. 27, 1997, now abandoned; which is a continuationof U.S. application Ser. No. 08/462,492, filed Jun. 5, 1995, now U.S.Pat. 5,716,832; which is a continuation of U.S. application Ser. No.08/156,759 filed Nov. 23, 1993, now U.S. Pat. No. 5,591,624; which is acontinuation of U.S. application Ser. No. 07/800,921, filed Nov. 27,1991, now abandoned; which is a continuation-in-part of U.S. applicationSer. No. 07/586,603, filed Sep. 21, 1990, now abandoned; which is acontinuation-in-part of U.S. application Ser. No. 07/565,606, filed Aug.10, 1990, now abandoned; which is a continuation-in-part of U.S.application Ser. No. 07/395,932, filed Aug. 18, 1989, now abandoned;which is a continuation-in-part of U.S. application Ser. No. 07/170,515,filed Mar. 21, 1988, now abandoned.

TECHNICAL FIELD

The present invention relates generally to retroviruses, and morespecifically, to recombinant retroviruses which are capable ofdelivering vector constructs to susceptible target cells. These vectorconstructs are typically designed to express desired proteins in targetcells, including proteins which can have a therapeutic effect in anumber of ways, and hence, constitute a “drug” transport system forallowing transport of proteins (or RNA) into cells. The specificity ofproteins (and RNA) for enzymatic reaction, for binding of cellularcomponents, for immunological action, or for other biological effects,allows for correspondingly specific actions on target cells if theprotein or RNA molecule can be transported into the cells. Such actionsinclude the repair of genetic defects, production of antisense RNA toblock cellular process, the enzymatic potentiation of prodrugs, andstimulation of the cellular immune system, as well as many othertherapies based on the intracellular production of proteins.

BACKGROUND OF THE INVENTION

Retroviruses are RNA viruses which can replicate and integrate into ahost cell's genome through a DNA intermediate. This DNA intermediate, orprovirus, may be stably integrated into the host's cellular DNA. Due totheir efficiency at integrating into host cells, retroviruses areconsidered to be one of the most promising vectors for use in human genetherapy. These vectors have a number of properties that lead them to beconsidered as one of the most promising techniques for genetic therapyof disease. These include: (1) efficient entry of genetic material (thevector genome) into cells; (2) an active efficient process of entry intothe target cell nucleus; (3) relatively high levels of gene expression;(4) minimal pathological effects on target cells; and (5) the potentialto target to particular cellular subtypes through control of thevector-target cell binding and the tissue-specific control of geneexpression. For example, a foreign gene of interest may be incorporatedinto the retrovirus in place of the normal retroviral RNA. When theretrovirus injects its RNA into a cell, the foreign gene is alsointroduced into the cell, and may then be integrated into the host'scellular DNA as if it were the retrovirus itself. Expression of thisforeign gene within the host results in expression of the foreignprotein by the host cell.

Most retroviruses which have been developed for gene therapy are murineretroviruses. Briefly, these retroviruses exist in two forms, asproviruses integrated into a host's cellular DNA, or as free virions.The virion form of the virus contains the structural and enzymaticproteins of the retrovirus (including reverse transcriptase), two RNAcopies of the viral genome, and portions of the cell's plasma membranein which is embedded the viral envelope glycoprotein. The genome isorganized into four main regions: the Long Terminal Repeat (LTR), andthe qag, pol, and env genes. The LTR may be found at both ends of theproviral genome, is a composite of the 5′ and 3′ ends of the RNA genome,and contains cis-acting elements necessary for the initiation andtermination of transcription. The three genes gag, pol, and env arelocated between the terminal LTRs. The gag and pol genes encode,respectively, internal viral structures and enzymatic proteins. The envgene encodes the envelope glycoprotein which confers infectivity andhost range specificity of the virus.

An important consideration in using retroviruses for gene therapy is theavailability of “safe” retroviruses. Packaging cell lines have beendeveloped to meet this concern. Briefly, this methodology employs theuse of two components, a retroviral vector and a packaging cell. Theretroviral vector contains long terminal repeats (LTRs), the foreign DNAto be transferred and a packaging sequence (ψ). This retroviral vectorwill not reproduce by itself because the genes which encode structuraland envelope proteins are not included within the vector. The packagingcell contains genes encoding the gag, pol, and env proteins, but doesnot contain the packaging signal “ψ”. Thus, a packaging cell can onlyform empty virion particles by itself. Within this general method, theretroviral vector is introduced into the packaging cell, therebycreating a “producer cell.” This producer cell manufactures virionparticles containing only the retroviral vector's (foreign) DNA, andtherefore has previously been considered to be a safe retrovirus fortherapeutic use.

There are several shortcomings in the current use of this approach. Oneissue involves the generation of “live virus” (i.e., competentreplicating retrovirus) by the producer cell line. Preparations of humantherapeutics which are contaminated with retroviruses are not currentlyconsidered suitable for use in human therapy. For example, extrememeasures are taken to exclude retroviral contamination of monoclonalantibodies for imaging and therapy. Live virus can in conventionalproducer cells when: (1) The vector genome and the helper genomesrecombine with each other; (2) The vector genome or helper genomerecombines with homologous cryptic endogenous retroviral elements in theproducer cell; or (3) Cryptic endogenous retroviral elements reactivate(e.g., xenotropic retroviruses in mouse cells).

Another issue is the propensity of mouse based producer lines to packageendogenous retroviral-vector-like elements (which can contain onc genesequences) at efficiencies close to that with which they package thedesired vector. Such elements, because of their vector-like structure,are transmitted to the target treatment cell at frequencies thatparallel its transfer of the desired vector sequence.

A third issue is the ability to make sufficient vector particles at asuitable concentration to: (1) treat a large number of cells (e.g.,10^(8–10) ¹⁰); and (2) manufacture vector particles at a commerciallyviable cost. Finally, the only producer lines currently used fortransfer of genes to human cells are amphotropic producer lines, knownfor the eponymous murine retroviral envelope gene, which has receptorsin most human cells.

In order to construct safer packaging cell lines, researchers havegenerated additional deletions in the 3′ LTR and portions of the 5′ LTR(see, Miller and Buttimore, Mol. Cell. Biol., 6:2895–2902, 1986). Whensuch cells are used, two recombination events are necessary to form thewild-type genome. Nevertheless, results from several laboratories haveindicated that even when several mutations are present, wild-type virusmay still be generated (see, Bosselman et al., Mol. Cell. Biol.7:1797–1806, 1987; Danos and Mulligan, Proc. Nat'l. Acad. Sci. USA81:6460–6464, 1988).

Many of the helper cell lines that have been described to date have beenlimited to a host cell range of murine, avian, rat and dog cells. whilelater helper cell lines have been generated using amphotropic retroviralvector systems, which can infect human cells as well as a broad range ofother mammalian cells (see, Sorge et al., Mol. Cell. Biol. 4:1720–1737,1984), amphotropic packaging lines developed thus far have retainedportions of one or more of the viral LTRs, and, thus, even when multiplemutations are present, have remained capable of generating areplication-competent genome. Amphotropic vector systems with multiplemutations and reduced propensities toward generating infectious virusgenerally exhibit unsatisfactorily low titres of retroviral particles.

One of the more recent approaches to constructing safer packaging celllines involves the use of complementary portions of helper virus,divided among two separate plasmids, one containing gag and pol, and theother containing env (see, Markowitz et al., J. Virol. 62:1120–1124; andMarkowitz et al., Virology 167: 600–606, 1988. One benefit of thisdouble-plasmid system is that three recombination events are required togenerate a replication competent genome. Nonetheless, thesedouble-plasmid vectors have also suffered from the drawback of includingportions of the retroviral LTRs, and therefore remain capable ofproducing infectious virus. Cell lines containing both 3′ and 5′ LTRdeletions have been constructed, but have thus far not proven usefulsince they produce relatively low titers (Daugherty et al., J. Virol.63:3209–3212, 1989).

The present invention overcomes difficulties of prior packaging celllines, and further provides other related advantages.

SUMMARY OF THE INVENTION

The present invention provides a method for producing recombinantretroviruses in which the retroviral genome is packaged in a capsid andenvelope, preferably through the use of a packaging cell. The packagingcells are provided with viral protein-coding sequences, preferably inthe form of two plasmids integrated into the genome of the cell, whichproduce all proteins necessary for production of viable retroviralparticles, a DNA viral construct which codes for an RNA which will carrythe desired gene, along with a packaging signal which will directpackaging of the RNA into the retroviral particles.

The present invention additionally provides a number of techniques forproducing recombinant retroviruses which can facilitate:

-   i) the production of higher titres from packaging cells;-   ii) the production of higher titres of helper free recombinant    retrovirus from packaging cell lines that are non-murine (to avoid    production of recombinant or endogenously activated retroviruses,    and to avoid packaging of defective murine retroviral sequences) and    which will infect human cells;-   iii) the production of helper free recombinant retroviruses with    higher titres using alternative non-hybrid envelopes such as    xenotropic or polytropic envelope proteins (to allow infection of    cells poorly infectable with amphotropic recombinant retroviruses or    to allow specificity of cell type infection).-   iv) packaging of vector constructs by means not involving the use of    packaging cells;-   v) the production of recombinant retroviruses which can be targeted    for preselected cell lines;-   vi) the construction of retroviral vectors with tissue-specific    (e.g., tumor) promoters; and-   vii) the integration of the proviral construct into a preselected    site or sites in a cell's genome.

One technique for producing higher titres from packaging cells takesadvantage of the discovery that of the many factors which can limittitre from a packaging cell, one of the most limiting is the level ofexpression of the packaging proteins, namely, the gag, pol, and envproteins, as well as the level of expression of the retroviral vectorRNA from the proviral vector. This technique allows the selection ofpackaging cells which have higher levels of expression (i.e., producehigher concentrations) of the foregoing packaging proteins and vectorconstruct RNA. More specifically, this technique allows selection ofpackaging cells which produce high levels of what is referred to hereinas a “primary agent,” which is either a packaging protein (e.g., gag,pol, or env proteins) or a gene of interest to be carried into thegenome of target cells (typically as a vector construct). This isaccomplished by providing in packaging cells a genome carrying a gene(the “primary gene”) which expresses the primary agent in the packagingcells, along with a selectable gene, preferably downstream from theprimary gene. The selectable gene expresses a selectable protein in thepackaging cells, preferably one which conveys resistance to an otherwisecytotoxic drug. The cells are then exposed to a selecting agent,preferably the cytotoxic drug, which enables identification of thosecells which express the selectable protein at a critical level (i.e., inthe case of a cytotoxic drug, by killing those cells which do notproduce a level of resistance protein required for survival).

Preferably, in the technique briefly described above, the expression ofboth the selectable and primary genes is controlled by the samepromoter. In this regard, it may be preferable to utilize a non-MLVretroviral 5′ LTR. In order to maximize titre of a recombinantretro-virus from packaging cells, this technique is first used to selectpackaging cells expressing high levels of all the required packagingproteins, and then is used to select which of these cells, followingtransfection with the desired proviral construct, produce the highesttitres of the recombinant retrovirus.

Techniques are also provided to select cells that produce higher titresof helper free recombinant retroviruses in non-murine cells. These celllines produce recombinant retroviruses capable of efficiently infectinghuman cells. These techniques involve screening potential parent cellsfor their ability to produce recombinant retroviruses in the presence ofa replicating virus. Subsequently, uninfected cultures of candidate celllines chosen by the above procedure are infected with a vectorexpressing a retroviral gag/pol, and clones which synthesize high levelsof gag/pol are identified. A clone of this type is then reinfected witha vector expressing env, and clones expressing high level of env (andgag/pol) are identified. Within the context of the present invention,“high levels” means discernibly greater than that seen in the standardmouse packaging line, PA317 on a Western blot analysis. Many non-mousecell lines such as human or dog have never been known to spontaneouslygenerate competent retrovirus, do not carry possible recombinationpartners for recombinant murine retroviral packaging or gene sequences;and do not carry genes which make RNA which may be packaged by the MLVsystem. Techniques are provided to generate cell lines which producehigh titres of recombinant retroviruses using alternative envelopes suchas xenotropic or polytropic by techniques similar to those describedabove. Such retroviruses may be used in infecting amphotropic resistantcells (xenotropic envelope) or infecting only a subset of cells(polytropic).

A technique suitable for producing recombinant retroviruses which can betargeted for preselected cell lines utilizes recombinant retroviruseshaving one or more of the following: an env gene comprised of acytoplasmic segment of a first retroviral phenotype, and anextracellular binding segment exogenous to the first retroviralphenotype (the binding segment being from a second viral phenotype orfrom another protein with desired binding properties which is selectedto be expressed as a peptide which will bind to the desired target);another viral envelope protein; another ligand molecule in place of thenormal envelope protein; or another ligand molecule along with anenvelope protein that does not lead to infection of the target celltype. Preferably, in the technique briefly described above, an env genecomprised of a cytoplasmic segment of a retroviral phenotype is combinedwith an exogenous gene encoding a protein having a receptor-bindingdomain to improve the ability of the recombinant retrovirus to bindspecifically to a targeted cell type, e.g., a tumor cell. In thisregard, it may be preferable to utilize a receptor-binding domain whichbinds to receptors expressed at high levels on the surface of the targetcell (e.g., growth factor receptors in tumor cells) or alternatively, areceptor-binding domain binding to receptors expressed at a relativelyhigher level in one tissue cell type (e.g., epithelial cells, ductalepithelial cells, etc., in breast cancer). One potential advantage totargeting with hybrid envelopes with specificity for growth factor oractivation receptors (like EGF or CD3 receptors is that binding of thevector itself may then lead to cell cycling, which is necessary forviral integration and expression. Within this technique, it may bepossible to improve and genetically alter recombinant retroviruses withspecificity for a given tumor by repeated passage of a replicatingrecombinant retrovirus in tumor cells; or by linking the vectorconstruct to a drug resistance gene and selecting for drug resistance.

Techniques for integrating a retroviral genome at a specific site in theDNA of a target cell involve the use of homologous recombination, oralternatively, the use of a modified integrase enzyme which willrecognize a specific site on the target cell genome. Such site specificinsertion allows genes to be inserted at sites on the target cells' DNA,which will minimize the chances of insertional mutagenesis, minimizeinterference from other sequences on the DNA, and allow insertion ofsequences at specific target sites so as to reduce or eliminate theexpression of an undesirable gene (such as a viral gene) in the DNA ofthe target cell.

It will be appreciated that any of the above-described techniques may beused independently of the others in particular situations, or can beused in conjunction with one or more of the remainder of the techniques.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts four plasmids designed to express retroviral proteins inmammalian cells. pSVgp and pRSVenv are cotransfected with a selectablemarker, while pSVgp-DHFR and pRSVenv-phleo are the equivalent plasmidswith the selectable marker placed downstream of the viral protein-codingregions.

FIG. 1B depicts vectors which lead to expression of: 1. MLV coreproteins (pSCV10); 2–4 MLV amphotropic env (pCMvenv AmDra, pCMVenvAnNhe, pMLPenv AmSph); 5. MLV xenotropic env (PCMV xeno). 6. MLV MCFenv(pCMV MCF); 7. MLV amphotropic env as a retroviral vector (pLARNL).

FIG. 1C depicts the results of the screening procedure for assessing theintrinsic ability of cell lines to make retroviral vectors in thepresence of helper virus (Example 2B).

FIG. 1D depicts the results of selecting clones of cells into whichpSCV10 had been introduced and examining these clones for gag productionas compared to PA317 by Western blots.

FIG. 1E depicts the results of Western blot experiments to comparelevels of amphotropic env in cell lysates from DA, CA, 2A and PA317.

FIG. 1F depicts the results of Western blot experiments to comparelevels of xenotropic env in cell lysates from transient transfections ofCF gag/pol and permanently expressing lines XF7, X6, X10 and PA317(ampho env).

FIG. 1G depicts the results of Western blot 10 experiments to comparelevels of MCF (polytropic) env in HT1080 derived clones, PA317(amphotropic env) and HX (xenotropic env).

FIG. 2 depicts three sites of fusion of HIV env and MoMLV env aftersite-directed mutagenesis. The joint at the extracellular margin of thetransmembrane region is designated as A, while B and C indicatelocations of joints at the middle of the transmembrane region andcytoplasmic margin, respectively. The numbering is according tonucleotide numbers (RNA Tumor Viruses, Vol. II, Cold Spring Harbor,1985). ST, SR, SE are the starts of tat, rev and env while TT, TR, andTE are the corresponding termination sites.

FIG. 3 depicts the substitution of U3 in a 5′ LTR by a heterologouspromoter/enhancer in which can be fused to either the Sac I, Bssh II orother site in the region.

FIG. 4 illustrates a representative method for crossing transgenic miceexpressing viral protein or vector RNA.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention is based, in part, upon thediscovery of the major causes of low recombinant virus titres frompackaging cell lines (PCL), and of techniques to correct those causes.Basically, at least five factors may be postulated as causes for lowrecombinant virus titres:

1. the limited availability of viral packaging proteins;

2. the limited availability of retroviral vector RNA genomes;

3. the limited availability of cell membrane for budding of therecombinant retroviruses;

4. the limited intrinsic packaging efficiency of the retroviral vectorgenome; and

5. the density of the receptor specific for the envelope of a givenretrovirus.

6. The limited availability of host cell constituents (such as RNA ormyristoylation, phosphorylation, glycosylation or proteolyticfunctions).

As noted above, the limited availability of viral packaging proteins isthe initial limiting factor in recombinant retrovirus production frompackaging cells. When the level of packaging protein in the packagingcells is increased, titre increases to about 10⁵ infectiousunits/milliliter, following which increasing packaging protein level hasno further effect on titres. However, titres can be further augmented byalso increasing the level of retroviral vector genome available forpackaging. Thus, as described herein, it is advantageous to selectproducer cells that manufacture the maximum levels of packaging proteinsand retroviral vector genomes. It has been discovered that the methodsof identifying, and thus selecting, packaging cells and producer cells,described earlier under the section entitled “Background of theInvention,” tend to lead to selection of many producer cells whichproduce low titres for the reasons described below.

The present invention takes advantage of the previously disadvantageousfact that the protein expression level of a gene downstream from the 5′LTR or other promoter, and spaced therefrom by an intervening gene, issubstantially less than if the intervening gene were absent. In thepresent invention, the selectable gene is placed downstream from a geneof the packaging genome or the gene of interest carried by the vectorconstruct, but is still transcribed under the control of the viral 5′LTR or other promoter without any splice donor or splice acceptor sites.This accomplishes two things. First, since the packaging genes or genesof interest are now upstream with no intervening gene between themselvesand the promoter, their corresponding proteins (packaging protein orprotein of interest) will be expressed at a higher level (five- totwentyfold) than the selectable protein. Second, the selectable proteinwill be expressed on average at a lower level, with the distribution oflevel of expression shifting toward lower levels. However, the selectionlevel for resistance to phleomycin remains the same, so that only thetop-end expressing cells survive. The levels of the packaging protein orof the protein of interest will still be proportional, only in thiscase, a higher level of selectable protein corresponds to a much higherlevel of packaging protein or protein of interest.

Preferably, the foregoing procedure is performed using a plasmidcarrying one of the proviral gag/pol or env packaging genes, along witha first selectable gene. These cells are then screened for the cellsproducing the highest levels of protein by reaction with an antibodyagainst gag/pol (or possibly env), a second enzyme or labelled antibody,and then sorted on a fluorescence-activated cell sorter (FACS) ordetected on a western blot. Alternatively, other tests for protein levelmay be used. Subsequently, the procedure and screening are repeatedusing those selected cells, and the other of the gag/pol or envpackaging genes. In this step, a second selectable gene (different fromthe first) would be required downstream from the packaging gene and thecells producing the largest amount of the of the second viral proteinselected. This cell line is a packaging cell line (PCL) that may be usedwith any available vector. The procedure and screening are then repeatedusing the surviving cells, with a plasmid carrying the proviral vectorconstruct bearing the gene of interest and a third selectable gene,different from the first or second selectable gene. As a result of thisprocedure, cells producing high titres of the desired recombinantretrovirus will be selected, and these can be cultured as required tosupply recombinant retrovirus. In addition, gag and pol can beindependently introduced and selected.

Example 1 describes the construction of gag/pol and env plasmidsdesigned to use these procedures.

EXAMPLE 1 Plasmids Designed to Make High Levels of Packaging Proteins(FIG. 1)

1. The 2.7 kb Xba I fragment from pPAM (Miller et al., Mol. Cell. Biol.5:431, 1985), which contains the amphotrophic env segment, was cloned inpUC18 at the Xba I site, then removed with Hind III and Sma I. Thisfragment was cloned into the vector pRSV neo (Gorman et al., Mol. Cell.Biol. 2:1044, 1982; Southern et al., J. Mol. Appl. Genet. 1:327, 1982)cut with Hind III and Pvu II, to give pRSV env. A 0.7 kb Bam HI to BstEII fragment from the plasmid pUT507 (Mulsant et al., Somat. Cell. Mol.Genet. 14:243, 1988) with the BstE II end filled in carries the phleoresistance coding sequence. The 4.2 kb Bam HI to Xho I fragment, thecontiguous 1.6 kb Xho I to Xba I (Xba I filled in) from RSVenv, and thephleo fragment were ligated to give pRSVenv-phleo.

2. A fragment from the Pst I site at nucleotide 563 of MLV (RNA TumorViruses, Vol. II, Cold Spring Harbor, 1985) to the Sca I site at 5870was derived from pMLV-K (Miller et al., 1985, op. cit.) and cloned inthe Pst I to Bam HI (Bam HI filled-in) fragment from p4aA8 (Jolly etal., Proc. Natl. Acad. Sci. USA 80:477, 1983) that has the SV40promoter, the pBR322 ampicillin resistance and origin of replication andthe SV40 poly A site. This gives pSVgp. pSVgpDHFR was made using thefollowing fragments:—the 3.6 kb Hind III to Sal I fragment from pSVgpcontaining the SV40 promoter plus MLV gag and some pol sequences; the2.1 kb Sal I to Sca I fragment from PMLV-K with the rest of the polgene, the 3.2 kb Xba I (Xba I filled-in) to Pst I fragment from pF400with the DHFR gene plus poly A site, pBR322 origin and half theampicillin resistance gene; the 0.7 kb Pst I to Hind III fragment frompBR322 with the other half of the ampicillin resistance gene. This givespSVgp-DHFR. All these constructs are shown in FIG. 1. These plasmids canbe transfected into 3T3 cells or other cells, and high levels of gag,pol or env obtained.

An additional method for accomplishing selection is to use a geneselection in one round and its antisense in a subsequent round. Forexample, gag/pol may be introduced into an HPRT-deficient cell with theHPRT gene and selected for the presence of this gene using that mediawhich requires HPRT for the salvage of purines. In the next round, theantisense to HPRT could be delivered downstream to env and the cellselected in 6 thioguanine for the HPRT-deficient phenotype. Largeamounts of antisense HPRT would be required in order to inactivate theHPRT gene transcripts, assuming no reversion occurred. A further methodof accomplishing selection is described below. Co-transfection of a 10×stoichiometric excess of the expression vector over the separateselectable marker ensures high copy number of expression vector in drugresistant cell clones. In most of the examples noted herein, the gag/poland envelope expression vectors were introduced independently (i.e.,separate transfections) so that the two structural genes would notrecombine or concatamerize (as transfected integrated DNA tends to do),assuring that the genes are unlinked in the genome. The steady-statelevel of intracellular MLV gag/pol and env was measured by proteinimmunoblotting. The relative ease, sensitivity, and reproducibility ofimmunoblotting allowed rapid, quantitative analysis of a large number ofcell clones necessary to identify over-expressors of the MLV structuralproteins (gag/pol in particular) (see Example 2).

In addition to the gag/pol expressing constructs which begin atnucleotide 563 of MOMLV, several others can be constructed which containupstream lead sequences. It has been observed by Prats et al. (RNA TumorViruses Meeting, Cold Spring Harbor, N.Y., 1988) that a glycosylatedform of the gag protein initiates at nucleotide 357 and a translationenhancer maps in the region between nucleotides 200–270. Therefore,gag/pol expressing constructs may be made beginning at the Bal I site(nucleotide 212) or Eag I site (nucleotide 346) to include theseupstream elements and enhance vector production. A preferred method ofaccomplishing this is to include degenerate mutations to inactivate thepackaging signal found here, without affecting the coding potential ofthe nucleic acid.

Envelope Substitutions

The ability to express gag/pol and env function separately allows formanipulation of these functions independently. A cell line thatexpresses ample amounts of gag/pol can be used, for example, to addressquestions of titre with regard to env. One factor resulting in lowtitres is the density of appropriate receptor molecules on the targetcell or tissue. A second factor is the affinity of the receptor for theviral envelope protein. Given that env expression is from a separateunit, a variety of envelope genes (requiring different receptorproteins), such as xenotropic, polytropic, or amphotrophic envs from avariety of sources, can be tested for highest titres on a specifictarget tissue.

Envelope proteins from one retrovirus can often substitute, to varyingdegrees, for that of another retrovirus. For instance, the envelope ofmurine virus 4070A, HTLV I, GALV, and BLV can each substitute for thatof MOMLV, albeit with a lower efficiency (Cone and Mulligan, Proc. Natl.Acad. Sci. USA 81:6349–53, 1984; Wilson et al., J. Virol. 63, 2374–78,1989; Ban et al., J. Gen. Virol. 70:1987–93, 1989). To increase thenumber of cell types that could be infected with MLV-based vectors, PCLswere generated which individually express either amphotropic,xenotropic, or polytropic envelopes. Vector produced from any of thesePCLs can be used to infect any cell which contains the correspondingdistinct receptor (Rein and Schultz, Virology 136:144–52, 1984). Somecell types may, for instance, lack the amphotropic receptor and thus beresistant to infection with amphotropic vector, but express thexenotropic receptor and therefore be infectable by xenotropic vector.One report suggests that xenotropic vector, in the presence ofreplication-complement xenotropic virus, may more effectively infecthuman hematopoietic progenitor cells (Eglitis et al., Biochem. Biophys.Res. Comm. 151:201–206, 1988). Xenotropic vector, in the presence ofreplication-competent xenotropic virus, also infects cells from otherspecies which are not easily infectable by amphotropic virus such asbovine, porcine, and equine (Delouis et al., Biochem. Biophys Res. Comm.169:80–14, 1990). The xenotropic PCLs will therefore be useful forveterinary purposes in these species. Another example would beutilization of the spleen focus-forming virus (SFFV) envelope gene whichmay allow targeting to cells containing the erythropoietin receptor (J.P. Li et al., Nature 343:762–764, 1990).

As a specific example, all of the amphotropic PCLs described herein(canine and human fibroblasts) were infectable by xenotropic vector butwere resistant to infection by amphotropic vector, presumably due to thephenomenon of “viral interference” (cf. A. Rein, Virology 120:251–57,1982). The xenotropic PCL therefore allows the facile infection of theseamphotropic PCLs, which in turn produces 10–100× higher titre than PCLswhose vector has been introduced by other means (Miller et al., Somat.Cell Mol. Genet. 12:175–83, 1986). In principle, a PCL expressing anyviral envelope which can function with the MLV vector and packagingsystem and whose corresponding cellular receptor is found in a givenPCL, is useful for allowing vector infection of that PCL.

Vector produced from the polytropic PCL described herein has a morerestricted host range on human cells than vector produced from eitheramphotropic or xenotropic PCLs (see data below). The polytropic PCL maytherefore be particularly useful for targeting vector to a specifichuman cell type. The reduced homology between both xenotropic andpolytropic envelopes with the MOMLV gag/pol and with the vector makesthese PCLs even less likely to generate replication-competent retrovirusby homologous recombination than amphotropic PCLs. Examples of the useof these methods are set forth below (see Example 2).

Furthermore, envelopes from nonmurine retrovirus sources can be used forpseudotyping a vector. The exact rules for pseudotyping (i.e., whichenvelope proteins will interact with the nascent vector particle at thecytoplasmic side of the cell membrane to give a viable viral particle(Tato, Virology 88:71, 1978) and which will not (Vana, Nature 336:36,1988), are not well characterized. However, since a piece of cellmembrane buds off to form the viral envelope, molecules normally in themembrane are carried along on the viral envelope. Thus, a number ofdifferent potential ligands can be put on the surface of viral vectorsby manipulating the cell line making gag and pol in which the vectorsare produced or choosing various types of cell lines with particularsurface markers. One type of surface marker that can be expressed inhelper cells and that can give a useful vector-cell interaction is thereceptor for another potentially pathogenic virus. The pathogenic virusdisplays on the infected cell surface its virally specific protein(e.g., env) that normally interacts with the cell surface marker orreceptor to give viral infection. This reverses the specificity of theinfection of the vector with respect to the potentially pathogenic virusby using the same viral protein-receptor interaction, but with thereceptors on the vector and the viral protein on the cell.

It may be desirable to include a gene which encodes for an irrelevantenvelope protein which does not lead to infection of target cells by thevector so produced, but does facilitate the formation of infectiousviral particles. For example, one could use human Sup T1 cells as ahelper line. This human T-cell line expresses CD4 molecules at highlevels on its surface. Conversion of this into a helper line can beachieved by expressing gag/pol with appropriate expression vectors andalso, if necessary, the Moloney ecotropic env gene product as anirrelevant (for human cells) envelope protein (the Moloney ecotropic envonly leads to infection of mouse cells). Vectors produced from such ahelper line would have CD4 molecules on their surfaces and therefore becapable of infecting only cells which express HIV env, such asHIV-infected cells.

In addition, hybrid envelopes (as described below) can be used in thissystem as well, to tailor the tropism (and effectively increase titres)of a retroviral vector. A cell line that expresses ample amounts of agiven envelope gene can be employed to address questions of titre withregard to gag and pol.

Furthermore, it is also possible to add ligand molecules exogenously tothe viral particles which either incorporate themselves in the lipidenvelope or can be linked chemically to the lipid or proteinconstituents.

Cell Lines

The most common packaging cell lines used for MOMLV vector systems(psi2, PA12, PA317) are derived from murine cell lines. There areseveral reasons why a murine cell line is not the most suitable forproduction of human therapeutic vectors:

1. They are known to contain endogenous retroviruses, some of which areclosely related in sequence and viral type to the MLV vector system usedhere.

2. They contain nonretroviral or defective retroviral sequences that areknown to package efficiently.

3. There may be deleterious effects caused by the presence of murinecell membrane components.

Several non-murine cell lines are potential parents for packaging lines.These include Vero cells which are used in Europe to prepare poliovaccine, WI38 which are used in the U.S. in vaccine production, CHOcells which are used in the U.S. for TPA preparation, D17 or other dogcells that may have no endogenous viruses, and those described inExample 2.

The most important safety concern for the production of retroviralvectors is the inherent propensity of retroviral PCLs to generatereplication-competent retrovirus after introduction of a vector (Munchauet al., Virology 176:262–65, 1990). This can occur in at least twoways: 1) homologous recombination can occur between the therapeuticproviral DNA and the DNA encoding the MOMLV structural genes (“gag/pol”and “env”) present in the PCL (discussed below under “Generation ofPCLs”); and 2) generation of replication-competent virus by homologousrecombination of the proviral DNA with the very large number ofdefective endogenous proviruses found in murine cells (Steffen andWeinberg, Cell 15:1003–10, 1978); Canaani and Aaronson, Proc. Natl.Acad. Sci., USA 76:1677–81, 1979; Stoye and Coffin, J. Virol. 61:2659–691987). In addition, even murine cell lines lacking vector can producevirus spontaneously or after induction, (e.g., xenotropic virus whichcan replicate in human cells, Aaronson and Dunn, J. Virol. 13:181–85,1974; Stephenson and Aaronson, Proc. Natl. Acad. Sci., USA 71:4925–29,1974; Aaronson and Stephenson, Biochem. Biophys. Acta 458:323–54, 1976).Another safety concern with the utilization of murine cells for theproduction of murine retroviral vectors is the observation that some ofthe many endogenous proviral genes (retrovirus-like genes) in the murinegenome are expressed, recognized by the retroviral structural geneproducts of murine PCLs, and delivered and expressed in target cellswith an efficiency at least comparable to that of the desired vector(Scolnick et al., J. Virol. 29:964–72, 1979; Scadden et al., J. Virol.64:424–27, 1990). These observations strongly suggest that murine celllines are an unsafe choice for the production of murine retroviralvectors for human therapeutics. To circumvent the inherent safetyproblems associated with murine cells, PCLs have been generatedexclusively from non-murine cell lines (e.g., canine and human celllines) which are known to lack genomic sequences homologous to that ofMOMLV by hybridization analysis (data not shown) (Martin et al., Proc.Natl. Acad. Sci., USA 78:4892–96, 1981). Those skilled in the art willrecognize that the packaging cells described herein will have a low, butinherent capability of packaging random RNA molecules. Such RNAmolecules will not be permanently transmitted to the pseudo-infectedtarget cell.

In addition to issues of safety, the choice of host cell line for thePCL is of importance because many of the physical (such as stability)and biological properties (such as titre) of retroviral particles aredictated by the properties of the host cell. For instance, the host cellmust efficiently express (transcribe) the vector RNA genome, prime thevector for first strand synthesis with a cellular tRNA, tolerate andcovalently modify the MLV structural proteins (proteolysis,glycosylation, myristylation, and phosphorylation), and the maturingvirion buds from the cell membrane, carrying many of the membranecomponents with it. For example, it has been found that vector made fromthe mouse packaging line PA317 is retained by a 0.3 micron filter, whilethat made from the CA line described herein will pass through.

EXAMPLE 2 Packaging Cell Selection

A. MLV Structural Gene Expression Vectors

To decrease the possibility of replication-competent virus beinggenerated by genetic interactions between the MLV proviral vector DNAand the structural genes of the PCL, separate expression vectors, eachlacking the viral LTR, were generated to express the gag/pol and envgenes independently. To further decrease the possibility of homologousrecombination with MLV vectors and the resultant generation ofreplication-competent virus, minimal sequences other than the proteincoding sequences were used. In order to express high levels of the MLVstructural proteins in the host cells, strong transcriptional promoters(CMV early and Ad5 major late promoters) were utilized. An example ofthe construction of a MoMLV gag/pol expression vector (pSCV10, see FIG.1B.1) follows:

1. The 0.7 Kb HinCII/XmaIII fragment encompassing the humancytomegalovirus (CMV) early transcriptional promoter (Boshart et al.,Cell 41:521–30, 1985) was isolated.

2. A 5.3 Kb PstI(partial)/ScaI fragment from the MoMLV proviral plasmid,MLV-K (Miller et al., Mol. Cell Biol. 5:531, 1985) encompassing theentire gag/pol coding region was isolated.

3. A 0.35 Kb DraI fragment from SV40 DNA (residues 2717–2363)encompassing the SV40 late transcriptional termination signal wasisolated.

4. Using linkers and other standard recombinant DNA techniques, the CMVpromoter-MoMLV gag/pol-SV40 termination signal was ligated into thebluescript vector SK⁺.

An example of the construction of an MLV amphotropic envelope expressionvector (pCMVenvAmDra, see FIG. 1B.2) follows.

1. A 2.7 Kb XbaI/NheI fragment containing the coding sequence ofamphotropic envelope from the 4070A proviral clone (Chattopadhyay etal., J. Virol. 39:777–91, 1981) was isolated.

2. Using linkers and other standard DNA techniques, the CMV earlypromoter and SV40 late termination signal described for the gag/polexpression above (pSCV10) were ligated in the order CMVpromoter-envelope-termination signal.

A second example of the construction of an MLV amphotropic envelopeexpression vector (PCMVenvAmNhe, see FIG. 1B.3) follows.

1. A 2.7 Kb XbaI/NheI fragment containing the coding sequence ofamphotropic envelope from the 4070A proviral clone described above wasisolated.

2. Using linkers and other standard recombinant DNA techniques, the CMVearly promoter described for the gag/pol expression above (pSCV10) wasligated in the plasmid pUC18 in the order CMV promoter-envelope (noadded transcriptional termination signal).

A third example of the construction of an MLV amphotropic envelopeexpression vector (pMLPenvAmSph, see FIG. 1B.4) follows.

1. A 0.9 Kb EcoRI/HindIII fragment containing the Adenovirus 5 left end,major late transcriptional promoter, and tripartite leader sequence wasisolated.

2. A 0.85 Kb EcoRI/BamHI fragment containing the SV40 small t intron andtranscriptional termination signal from clone pJD204 (De Wit et al.,Mol. Cell. Biol. 7:725–37, 1987) was isolated.

3. A 3 Kb SphI/SmaI fragment containing the coding sequence ofamphotropic envelope from the 4070A proviral clone described above wasisolated.

4. Using linkers and other standard recombinant DNA techniques, the MLP,amphotropic envelope and the SV40 termination signal were ligated inplasmid pBR322 in the order MLP-envelope-SV40.

An example of the construction of an MLV xenotropic envelope expressionvector (pCMMVxeno, see FIG. 1B.5) follows.

1. A 2.2 Kb NaeI/NheI fragment containing the coding region of thexenotropic envelope obtained from clone NZB9–1 (O'Neill et al., J.Virol. 53:100–106, 1985) was isolated.

2. Using linkers and other standard recombinant DNA techniques, the CMVearly promoter and SV40 late termination signal described for thegag/pol expression above (pSCV10) were ligated in the order CMVpromoter-envelope-termination signal.

An example of the construction of an MLV polytropic envelope expressionvector (pCMVMCF, see FIG. 1B.6) follows.

1. A 2 Kb BamHI/NheI fragment containing the coding region of thepolytropic envelope obtained from clone MCF-247W (Holland et al., J.Virol. 53:152–57, 1985) was isolated.

2. Using linkers and other standard recombinant DNA techniques, the CMVearly promoter and SV40 late termination signal described for thegag/pol expression above (pSCV10) were ligated in the order CMVpromoter-envelope-termination signal.

An example of the construction of an MLV ampho env Neo⁺ retroviralvector (pLARNL, FIG. 1B.7) follows.

1. The vector PLRNL vector (Emi et al., J. Virol. 65:1202–1207, 1991)was digested with BamHI.

2. A 2.7 Kb XbaI fragment containing the envelope protein coding regionof retrovirus 4070A (Chattopadhyay et al., J. Virol. 39:777–91, 1981)was isolated.

3. Fragments from procedures 1 and 2 above were ligated.

B. Host Cell Selection

Host cell lines were screened for their ability to efficiently (hightitre) rescue a drug resistance retroviral vector (A alpha N2) usingreplication competent retrovirus to produce the gag/pol and envstructural genes (“MA” virus). Titre was measured from confluentmonolayers 16 h after a medium change by adding filtered supernatants(0.45 um filters) to 5×10⁴ NIH 3T3 TK⁻ cells on a 6 cm tissue cultureplate in the presence of 4 ug/ml polybrene followed by selection inG418.

Data from the screening process is shown in FIG. 2. Among the non-murinecell lines which demonstrate the ability to package MoMLV-based vectorwith high titre are the cell lines CF2, D17, 293, and HT1080. These celllines were used herein as examples, although any other cells may betested by such means.

C. Generation of Packaging Cell

(i) gag/pol Intermediate

As examples of the generation of gag/pol intermediates for PCLproduction, D17, 293, and HT1080 were co-transfected with 1 ug of themethotrexate resistance vector, pFR400 (Graham and van der Eb, Virology52:456–67, 1973), and 10 ug of the MOMLV gag/pol expression vector,pSCV10 (above) by calcium phosphate co-precipitation (D17 and HT1080,see Graham and van der Eb, Virology 52:456–67, 1973), or lipofection(293, see Felgner et al., Proc. Natl. Acad. Sci., USA 84:7413–17, 1987).After selection for transfected cells in the presence of the drugsdipyrimidol and methotrexate, individual drug resistant cell colonieswere expanded and analyzed for MOMLV gag/pol expression by extracellularreverse transcriptase (RT) activity (modified from Goff et al., J.Virol. 38:239–48, 1981) and intracellular p30^(gag) by western blotusing anti p30 antibodies (goat antiserum #77S000087 from the NationalCancer Institute). This method identified individual cell clones in eachcell type which expressed 10–50× higher levels of both proteins comparedwith that of a standard mouse amphotropic PCL, PA317 (FIG. 1D and Table1).

TABLE 1 PROPERTIES OF MoMLV GAG/POL-EXPRESSING CELLS LARNL RT p30^(gag)TITRE CELL NAME ACTIVITY (CPM) EXPRESSION (CFU/ML) 3T3 800 − N.D. PA3171350 +/− 1.2 × 10³ D17 800 − N.D. D17 4–15 5000 +++++ 1.2 × 10⁴ D17 9–202000 +++ 6.0 × 10³ D17 9–9 2200 ++ 1.0 × 10³ D17 9–16 6100 +++++ 1.5 ×10⁴ D17 8–7 4000 − N.D. HT1080 900 − N.D. HTSCV21 16400 +++++ 8.2 × 10³HTSCV25 7900 +++ 2.8 × 10³ HTSCV42 11600 ++ 8.0 × 10² HTSCV26 4000 − <10293 600 − N.D. 293 2–3 6500 +++++   7 × 10⁴ 293 5–2 7600 +++++ N.D.

The biological activity of these proteins was tested by introducing aretroviral vector, LARNL (see FIG. 1B) which expresses both theamphotropic envelope and a Neo⁺ marker which confers resistance to thedrug, G418. In every case, co-expression of gag/pol in the cell line andenv from the vector allowed efficient packaging of the vector asdetermined by cell-free transfer of G418 resistance to 3T3 cells(titre). Titre was measured from confluent monolayers 16 h after amedium change by adding filtered supernatants (0.45 um filters) to 5×10⁴NIH3T3 TK⁻ cells on a 6 cm tissue culture plate in the presence of 4ug/ml polybrene followed by selection in G418. Significantly, the vectortitres from the cell lines correlated with the levels of p30^(gag)(Table 1). Since the level of env should be the same in each clone andis comparable to the level found in PA317 (data not shown), thisindicates that titre was limited by the lower levels of gag/pol in thesecells (including PA317). The titre correlated more closely with thelevels of p30^(gag) than with the levels of RT.

(ii) Conversion of gag/pol Lines into Amphotropic Packaging Lines

As examples of the generation of amphotropic PCLs, the gag/polover-expressors for 293 (termed 2–3) and D17 (termed 4–15) wereco-transfected by the same techniques described above except that 1 ugof the phleomycin resistance vector, pUT507 (Mulsant et al., Somat. CellMol. Genet. 14:243–52, 1988), and 10 ug of the amphotropic envelopeexpression vectors, pMLPenvAmSph (for 2–3) or pCMVenvAmNhe (for 4–15)were used. After selection for transfected cells in the presence ofphleomycin, individual drug resistant cell colonies were expanded andanalyzed for intracellular gp80^(env) expression by western blot usinganti gp70 (goat antiserum #79S000771 from N.C.I.). Several clones wereidentified which expressed relatively high levels of both gag/pol andampho env (PCLs, see FIG. 1 for representative data).

In another example of the generation of an ampho PCL, CF2 cells wereelectroporated (cf. Chu et al., Nucl. Acids Res. 15:1311–26, 1987) with2 ug of the phleomycin resistance marker, pUT507, 10 ug of pSCV10(above), and 10 ug of pCMVenvAmNhe (above). After selection fortransfected cells in the presence of phleomycin, individual drugresistant cell colonies were expanded and analyzed for intracellularexpression of MLV p30^(gag) and gp80^(env) proteins by western blotusing specific antisera. A clone was identified which expressedrelatively high levels of both gag/pol and ampho env (FIG. 1E).

(iii) Performance of Amphotropic Packaging Cell Lines

A number of these ampho PCLs were tested for their capacity to packageretroviral vectors by measuring titre after the introduction ofretroviral vectors (Table 2). The measurements were performed usinguncloned PCLs, so that the average performance of the lines wascalculated.

TABLE 2 VECTOR TITRE AND HELPER VIRUS GENERATION IN AMPHOTROPIC PCLsVECTOR TITRE^(a) (+/− HELPER VIRUS^(b)) CELL TYPE b-Gal KT-1 N2 PA3173.5 × 10² (N.D.) 1.0 × 10⁴ (N.D.) 3.0 × 10⁵(+)^(c) CA 5.0 × 10⁴ (N.D.)3.0 × 10⁵(−)^(d) 2.0 × 10⁶(−)^(d) 2A 4.0 × 10⁴ (N.D.) 2.0 × 10⁵(−)^(e)N.D. DA N.D. N.D. 2.0 × 10⁵(−)^(d) DA2 N.D. 3.9 × 10⁵(−)^(d) N.D.^(a)cfu/ml ^(b)as judged by marker rescue assay with MA virus aspositive control ^(c)after 20 days in culture ^(d)after 60 days inculture ^(e)after 90 days in culture

Highest titres are obtained when retroviral vectors were introduced intoPCLs by infection (Miller et al., Somat. Cell Mol. Genet. 12:175–83,1986). However, although amphotropic MLV vectors are known to infectthese host cell types, the PCLs are blocked for infection by amphovector since they express ampho env (“viral interference”). To overcomethis problem, vectors containing other viral envelopes (such asxenotropic env or VSV G protein, which bind to cell receptors other thanthe ampho receptor) were generated in the following manner. Ten ug ofthe vector DNA of interest was co-transfected with 10 ug of DNA whichexpresses either xeno env (pCXvxeno, above) or a VSV G proteinexpression vector, MLP G, onto a cell line which expresses high levelsof MOMLV gag/pol such as 2–3 cell (see above). The resultant vectorcontaining xenotropic env or VSV G protein, respectively, was producedtransiently in the co-transfected cells and after 2 days cell freesupernatants were added to the potential PCLs, and vector-infected cellswere identified by selection in G418. Both types of vector efficientlyinfected the ampho-blocked cells and after G418 selection cell freesupernatants were collected from the confluent monolayers and titred onNIH 3T3 TK⁻ cells as described above. The cell clones with the highesttitre were chosen as PCLs and referred to as DA (D17 ampho), 2A (293ampho), and CA (CF2 ampho), respectively. In no case was helper virusdetected in the currently described PCLs, even when a retroviral vector(N2) which has a high probability of generating helper virus (Armentanoet al., J. Virol. 62:1647–50, 1987) was introduced into the PCLs and thecells passaged for as long as 2 months (3 months for vector KT-3). Onthe other hand, the same vector introduced into the PA317 cell linegenerated helper virus within 3 weeks of continual passaging.

(iv) Conversion of gag/pol Lines into Xenotropic Packaging Cell Lines

As examples of the generation of xenotropic PCLS, the gag/polover-expressors for D17 (4–15) and HT1080 (SCV21) were co-transfected bythe same techniques described above except that 1 ug of either thephleomycin resistance vector, pUT507 (for SCV21), or the hygromycin Bresistance marker, pY3 (for 4–15, see Blochlinger and Diggelmann, Mol.Cell Biol. 4:2929–31, 1984), and 10 ug of the xenotropic envelopeexpression vector, pCMVxeno (above) was used. After selection fortransfected cells in the presence of phleomycin or hygromycin,respectively, individual drug resistant cell colonies were expanded andanalyzed for intracellular expression of MLV p30^(gag) and gp75^(env)proteins by western blot using specific antisera. Clones were identifiedwhich expressed relatively high levels of both gag/pol and xeno env(FIG. 1F).

(v) Performance of Xenotropic Packaging Cell Lines

A number of these potential xeno PCLs were tested for their capacity topackage retroviral vectors by measuring titre after the introduction ofretroviral vectors (Table 3).

TABLE 3 VECTOR TITRE ON XENOTROPIC PCLs KT-1 TITRE (CFU/ML) CELL CLONEON HT1080 CELLS HT1080 SCV21 XF1 1.0 × 10⁵ XF7 1.0 × 10⁵ XF12 (HX) 4.5 ×10⁵ D17 4–15 X6 9.0 × 10⁴ X10 (DX) 1.3 × 10⁵ X23 8.0 × 10⁴

As described above, vector containing VSV G protein was producedtransiently in 2–3 cells. After 2 days, cell free supernatants wereadded to the xeno PCLs and after G418 selection cell free supernatantswere collected from the confluent monolayers and titred as describedabove except that HT1080 cells, which are infectable by xeno vector, wasused instead of NIH 3T3 TK⁻ cells which are resistant to xeno vector.The cell clones with the highest titre were chosen as PCLs and referredto as DX (D17 xeno) and HX (HT1080 xeno), respectively.

The propensity of the PCLs described above to generate helper virus wastested even more stringently by co-cultivating ampho and xeno PCLscontaining the vector, N2. Since ampho vector can infect the xeno PCLsand vice versa, this allows continuous cross-infection events, each ofwhich increases the probability of generating helper virus. As anexample, 2A cells containing N2 were co-cultivated with HX cellscontaining N2. After 23 days, the cultures were still free of ampho andxeno viruses as judged by a vector rescue assay on 293 or Mus dunnicells, both of which can detect ampho and xeno viruses (Table 4).

TABLE 4 HIGH STRINGENCY ANALYSIS FOR PCL TENDENCY TO GENERATE HELPERVIRUS TEST MATERIAL HELPER VIRUS ASSAY AMPHOTROPIC VIRUS + XENOTROPICVIRUS + PA317 + N2 (21d) + 2A + HX + N2 (23d) −

(vi) Conversion of gag/pol Lines into Polytropic Packaging Cell Lines

As an example of the generation of a polytropic PCL, the gag/polover-expressor for HT1080 (SCV21) was co-transfected by the sametechniques described above, except that 1 ug of the phleomycinresistance vector, pUT507, and 10 ug of the polytropic envelopeexpression vector, pCMVMCF (above) was used. After selection fortransfected cells in the presence of phleomycin, individual drugresistant cell colonies were expanded and analyzed for intracellularexpression of MLV gp70^(env) protein by western blot using specificantiserum. Clones were identified which expressed relatively high levelsof both gag/pol (not shown) and polytropic env (FIG. 1G).

(vii) Performance of Polytropic Packaging Cell Lines

One of these potential poly PCLs (clone 3) was tested for the capacityto package retroviral vectors by measuring titre after the introductionof retroviral vectors (Table 5).

TABLE 5 HOST-RANGE OF POLYTROPIC VECTOR FROM HP CELLS CELL LINE SPECIESβ-Gal TITRE 3T3 MURINE 1.0 × 10⁴ PA317 MURINE 1.0 × 10⁴ 208F/C5 RAT 4.0× 10⁴ Mv-1-Lu MINK 5.0 × 10³ FRhL MACAQUE <10 HT1080 HUMAN <10 HeLaHUMAN <10 WI 38 HUMAN <10 DETROIT 551 HUMAN <10 SUP TI HUMAN <10 CEMHUMAN <10 U937 HUMAN <10 293 HUMAN 2.0 × 10⁴ AAT HUMAN <10 VandenbergHUMAN <10

This cell clone was chosen as PCL and referred to as HP (HT1080 poly).As described above, vector containing VSV G protein was producedtransiently in 2–3 cells and after 26 days, cell free supernatants wereadded to the polytropic PCL (HP). After G418 selection, cell freesupernatants were collected from the confluent monolayers and titred asdescribed above on a variety of cell lines. The infection of human cellswas very restricted, with all cell lines tested being negative with theexception of 293 cells.

Although the factors that lead to efficient infection of specific celltypes by retroviral vectors are not completely understood, it is clearthat because of their relatively high mutation rate, retroviruses may beadapted for markedly improved growth in cell types in which initialgrowth is poor, simply by continual reinfection and growth of the virusin that cell type (the adapter cell). This can also be achieved usingviral vectors that encode some viral functions (e.g., env), and whichare passed continuously in cells of a particular type which have beenengineered to have the functions necessary to complement those of thevector to give out infectious vector particles (e.g., gag/pol). Forexample, one can adapt the murine amphotropic virus 4070A to humanT-cells or monocytes by continuous growth and reinfection of eitherprimary cell cultures or permanent cell lines such as Sup T1 (T-cells)or U937 (monocytes). Once maximal growth has been achieved, as measuredby reverse transcriptase levels or other assays of virus production, thevirus is cloned out by any of a number of standard methods, the clone ischecked for activity (i.e., the ability to give the same maximal growthcharacteristic on transfection into the adapter cell type) and thisgenome used to make defective helper genomes and/or vectors which inturn, in an appropriately manufactured helper or producer line, willlead to production of viral vector particles which infect and express inthe adapter cell type with high efficiency (10⁷–10⁹ infectiousunits/ml).

VII. Alternative Viral Vector Packaging Techniques

Two additional alternative systems can be used to produce recombinantretroviruses carrying the vector construct. Each of these systems takesadvantage of the fact that the insect virus, baculovirus, and themammalian viruses, vaccinia and adenovirus, have been adapted recentlyto make large amounts of any given protein for which the gene has beencloned. For example, see Smith et al. (Mol. Cell. Biol. 3:12, 1983);Piccini et al. (Meth. Enzymology, 153:545, 1987); and Mansour et al.(Proc. Natl. Acad. Sci. USA 82:1359, 1985).

These viral vectors can be used to produce proteins in tissue culturecells by insertion of appropriate genes into the viral vector and,hence, could be adapted to make retroviral vector particles.

Adenovirus vectors are derived from nuclear replicating viruses and canbe defective. Genes can be inserted into vectors and used to expressproteins in mammalian cells either by in vitro construction (Ballay etal., EMBO J. 4:3861, 1985) or by recombination in cells (Thummel et al.,J. Mol. Appl. Genetics 1:435, 1982).

One preferred method is to construct plasmids using the adenovirus MajorLate Promoter (MLP) driving: (1) gag/pol, (2) env, (3) a modified viralvector construct. A modified viral vector construct is possible becausethe U3 region of the 5′ LTR, which contains the viral vector promoter,can be replaced by other promoter sequences (see, for example, Hartman,Nucl. Acids Res. 16:9345, 1988). This portion will be replaced after oneround of reverse transcriptase by the U3 from the 3′ LTR.

These plasmids can then be used to make adenovirus genomes in vitro(Ballay et al., op. cit.), and these transfected in 293 cells (a humancell line making adenovirus E1A protein), for which the adenoviralvectors are defective, to yield pure stocks of gag/pol, env andretroviral vector carried separately in defective adenovirus vectors.Since the titres of such vectors are typically 10⁷–10¹¹/ml, these stockscan be used to infect tissue culture cells simultaneously at highmultiplicity. The cells will then be programmed to produce retroviralproteins and retroviral vector genomes at high levels. Since theadenovirus vectors are defective, no large amounts of direct cell lysiswill occur and retroviral vectors can be harvested from the cellsupernatants.

Other viral vectors such as those derived from unrelated retroviralvectors (e.g., RSV, MMTV or HIV) can be used in the same manner togenerate vectors from primary cells. In one embodiment, these adenoviralvectors are used in conjunction with primary cells, giving rise toretroviral vector preparations from primary cells.

In some cases, gene products from other viruses may be used to improvethe properties of retroviral packaging systems. For instance, HIV revprotein might be included to prevent splicing of HIV env or HIV gag/polMLV vectors or HIV sor might increase the infectivity of T cells by freevirus as it does with HIV (See Fischer et al., Science 237:888–893,1987).

In an alternative system (which is more truly extracellular), thefollowing components are used:

1. gag/pol and env proteins made in the baculovirus system in a similarmanner as described in Smith et al. (supra) (or in other proteinproduction systems, such as yeast or E. coli);

2. viral vector RNA made in the known T7 or SP6 or other in vitroRNA-generating system (see, for example, Flamant and Sorge, J. Virol.62:1827, 1988);

3. tRNA made as in (2) or purified from yeast or mammalian tissueculture cells;

4. liposomes (with embedded env protein); and

5. cell extract or purified necessary components (when identified)(typically from mouse cells) to provide env processing, and any or othernecessary cell-derived functions.

Within this procedure (1), (2) and (3) are mixed, and then env protein,cell extract and pre-liposome mix (lipid in a suitable solvent) added.It may, however, be necessary to earlier embed the env protein in theliposomes prior to adding the resulting liposome-embedded env to themixture of (1), (2), and (3). The mix is treated (e.g., by sonication,temperature manipulation, or rotary dialysis) to allow encapsidation ofthe nascent viral particles with lipid plus embedded env protein in amanner similar to that for liposome encapsidation of pharmaceuticals, asdescribed in Gould-Fogerite et al., Anal. Biochem. 148:15, 1985). Thisprocedure allows the production of high titres of replicationincompetent recombinant retroviruses without contamination withpathogenic retroviruses or replication-competent retroviruses.

VIII. Cell Line-Specific Retroviruses—“Hybrid Envelope”

The host cell range specificity of a retrovirus is determined in part bythe env gene products. For example, Coffin, J. (RNA Tumor Viruses2:25–27, Cold Spring Harbor, 1985) notes that the extracellularcomponent of the proteins from murine leukemia virus (MLV) and RousSarcoma virus (RSV) are responsible for specific receptor binding. Thecytoplasmic domain of envelope proteins, on the other hand, areunderstood to play a role in virion formation. While pseudotyping (i.e.,the encapsidation of viral RNA from one species by viral proteins ofanother species) does occur at a low frequency, the envelope protein hassome specificity for virion formation of a given retrovirus. The presentinvention recognizes that by creating a hybrid env gene product (i.e.,specifically, an env protein having cytoplasmic regions and exogenousbinding regions which are not in the same protein molecule in nature)the host range specificity may be changed independently from thecytoplasmic function. Thus, recombinant retroviruses can be producedwhich will specifically bind to preselected target cells.

In order to make a hybrid protein in which the receptor bindingcomponent and the cytoplasmic component are from different retroviruses,a preferred location for recombination is within the membrane-spanningregion of the cytoplasmic component. Example 10 describes theconstruction of a hybrid env gene which expresses a protein with the CD4binding portion of the HIV envelope protein coupled to the cytoplasmicdomain of the MLV envelope protein.

EXAMPLE 3 Hybrid HIV-MLV Envelopes

A hybrid envelope gene is prepared using in vitro mutagenesis (Kunkel,Proc. Natl. Acad. Sci. USA 82:488–492, 1985) to introduce a newrestriction site at an appropriate point of junction. Alternatively, ifthe two envelope sequences are on the same plasmid, they can be joineddirectly at any desired point using in vitro mutagenesis. The end resultin either case is a hybrid gene containing the 5′ end of the HIV gp 160and the 3′ end of MLV p15E. The hybrid protein expressed by theresulting recombinant gene is illustrated in FIG. 2 and contains the HIVgp120 (CD4 receptor binding protein), the extracellular portion of HIVgp 41 (the gp 120 binding and fusigenic regions), and the cytoplasmicportion of MLV p15E, with the joint occurring at any of several pointswithin the host membrane. A hybrid with a fusion joint at thecytoplasmic surface (joint C in FIG. 2) causes syncytia when expressedin Sup T1 cells. The number of apparent syncytia are approximatelyone-fifth that of the nonhybrid HIV envelope gene in the same expressionvector. Syncytia with the hybrid occurs only when the rev protein isco-expressed in trans. A hybrid with a fusion joint at the extracellularsurface (joint A in FIG. 2) gives no syncytia while hybrid B (in themiddle of transmembrane regions) gives approximately five-fold lesssyncytium on Sup T1 cells than hybrid C.

While Example 3 illustrates one hybrid protein produced from twodifferent retroviruses, the possibilities are not limited toretroviruses or other viruses. For example, the receptor binding portionof human interleukin-2 may be combined with the envelope protein of MLVto target vectors to cells with IL-2 receptors. In this case, arecombination would preferably be located in the gp 70 portion of theMLV env gene, leaving an intact p15E protein. Furthermore, the foregoingtechnique may be used to create a recombinant retrovirus with anenvelope protein which recognizes antibody Fc segments. Monoclonalantibodies which recognize only preselected target cells only could thenbe bound to such a recombinant retrovirus exhibiting such envelopeproteins so that the retrovirus would bind to and infect only thosepreselected target cells. Alternatively, a hybrid envelope with thebinding domain of avidin would be useful for targeting cells' “images”in a patient or animal with biotinylated antibodies or other ligands.The patient would first be flooded with antibodies, and then antibodybinding nonspecifically allowed to clear from the patient's system,before administering the vector. The high affinity (10⁻¹⁵) of the avidinbinding site for biotin would then allow accurate and efficienttargeting to the original tissue identified by the monoclonal “image.”

The approach may also be used to achieve tumor-specific targeting andkilling by taking advantage of three levels of retroviral vectorspecificity; namely, cell entry, gene expression, and choice of proteinexpressed. Retroviral vectors enter cells and exert their effects atintracellular sites. In this respect their action is quite unique. Usingthis property, and the three levels of natural retroviral specificity(above), retroviral vectors may be engineered to target and kill tumorcells.

The overall goal of targeting of retrovirus to tumor cells may beaccomplished by two major experimental routes; namely, a) selection intissue culture (or in animals) for retrovituses that grow preferentiallyin tumor cells; or b) construction of retroviral vectors with tissue(tumor)-specific promoters with improvements being made by in vitropassage, and negative and positive drug-sensitivity selection.

Vectors suitable for selectively infecting selected cell types, such asa tumor cell, may generally be prepared by (a) continuously passaging avirus in cells of the selected cell type until the virus has geneticallymutated and a predominant fast growing strain has evolved; (b) isolatingthe mutated and fast growing strain; (c) identifying and isolating thecomponents of the mutated strain responsible for the preferential growthof the mutated virus; (d) inserting the identified and isolatedcomponents as substitutes for counterpart components in a producer cellbased upon the virus (prior to continuous passage); and (e) culturingthe producer cell to produce the vector.

At least four selective protocols may be utilized to select forretrovirus which grow preferentially in tumor cells; namely, 1) “EnvSelection by Passage In Vitro,” wherein selection of retrovirus withimproved replicative growth ability is accomplished by repeated passagein tumor cells; 2) “Selection with a Drug Resistance Gene,” whereingenetic selection for tumor “specific” retroviruses is based on viralconstructs containing a linked drug resistance gene; 3) “Hybrid-Env,”wherein selection (by protocol #1 or #2, above) of retrovirus withtumor-“specificity” is initiated from a construct containing a hybridenvelope gene which is a fusion of a tumor receptor gene (i.e., ananti-tumor antibody H-chain V-region gene fused with env; or, a growthreceptor fused with env); in this case selection begins at a favorablestarting point, e.g., an env which has some specificity for tumor cells;or 4) “Selection by Passage In Vitro and Counter Selection byCo-cultivation with Normal Cells,” wherein growth in tumor cells isselected-for by repeated passage in mixtures of drug-resistant tumorcells and drug-sensitive normal cells.

With respect to retroviral vector constructs carrying tissue(tumor)-specific promoters, biochemical markers with different levels oftissue-specificity are well known, and genetic control throughtissue-specific promoters is understood in some systems. There are anumber of genes whose transcriptional promoter elements are relativelyactive in rapidly growing cells (i.e., transferring receptor, thymidinekinase, etc.) and others whose promoter/enhancer elements are tissuespecific (i.e., HBV enhancer for liver, PSA promoter for prostate).Retroviral vectors and tissue-specific promoters (present either as aninternal promoter or within the LTR) which can drive the expression ofselectable markers and cell cycle genes (i.e., drug sensitivity, Ecogpt; or HSVTK in TK-cells). Expression of these genes can be selectedfor in media containing mycophenolic acid or HAT, respectively. In thismanner, tumor cells containing integrated provirus which activelyexpresses the drug resistance gene will survive. Selection in thissystem may involve selection for both tissue-specific promoters andviral LTRs. Alternatively, specific expression in tumor cells, and notin normal cells, can be counter-selected by periodically passaging virusonto normal cells, and selecting against virus that express Eco gpt orHSVtk (drug sensitivity) in those cells (by thioxanthine or acyclovir).Infected cells containing integrated provirus which express Eco gpt ortk phenotype will die and thus virus in that cell type will be selectedagainst.

IX. Site-Specific Integration

Targeting a retroviral vector to a predetermined locus on a chromosomeincreases the benefits of gene-delivery systems. A measure of safety isgained by direct integration to a “safe” spot on a chromosome, i.e., onethat is proven to have no deleterious effects from the insertion of avector. Another potential benefit is the ability to direct a gene to an“open” region of a chromosome, where its expression would be maximized.Two techniques for integrating retroviruses at specific sites aredescribed below.

(ii) Integrase Modification

Another technique for integrating a vector construct into specific,preselected sites of a target cell's genome involves integrasemodification.

The retrovirus pol gene product is generally processed into four parts:(i) a protease which processes the viral gag and pol products; (ii) thereverse transcriptase; and (iii) RNase H, which degrades RNA of anRNA/DNA duplex; and (iv) the endonuclease or “integrase.”

The general integrase structure has been analyzed by Johnson et al.(Proc. Natl. Acad. Sci. USA 83:7648–7652, 1986). It has been proposedthat this protein has a zinc binding finger with which it interacts withthe host DNA before integrating the retroviral sequences.

In other proteins, such “fingers” allow the protein to bind to DNA atparticular sequences. One illustrative example is the steroid receptors.In this case, one can make the estrogen receptor, responding toestrogens, have the effect of a glucocorticoid receptor, responding toglucocorticoids, simply by substituting the glucocorticoid receptor“finger” (i.e., DNA binding segment) in place of the estrogen receptorfinger segment in the estrogen receptor gene. In this example, theposition in the genome to which the proteins are targeted has beenchanged. Such directing sequences can also be substituted into theintegrase gene in place of the present zinc finger. For instance, thesegment coding for the DNA binding region of the human estrogen receptorgene may be substituted in place of the DNA binding region of theintegrase in a packaging genome. Initially, specific integration wouldbe tested by means of an in vitro integration system (Brown et al., Cell29:347–356, 1987). To confirm that the specificity would be seen invivo, this packaging genome is used to make infectious vector particles,and infection of and integration into estrogen-sensitive andestrogen-nonsensitive cells compared in culture.

Through use of this technique, incoming viral vectors may be directed tointegrate into preselected sites on the target cell's genome, dictatedby the genome-binding properties of site-specific DNA-bindingprotein-segments spliced into the integrase genome. It will beunderstood by those skilled in the art that the integration site must,in fact, be receptive to the fingers of the modified integrase. Forexample, most cells are sensitive to glucocorticoids and hence theirchromatin has sites for glucocorticoid receptors. Thus, for most cells,a modified integrase having a glucocorticoid receptor finger would besuitable to integrate the proviral vector construct at thoseglucocorticoid receptor-binding sites.

X. Production of Recombinant Retroviral Vectors in Transgenic Animals

Two problems previously described with helper line generation ofretroviral vectors are: (a) difficulty in generating large quantities ofvectors; and (b) the current need to use permanent instead of primarycells to make vectors. These problems can be overcome with producer orpackaging lines that are generated in transgenic animals. These animalswould carry the packaging genomes and retroviral vector genomes. Currenttechnology does not allow the generation of packaging cell lines anddesired vector-producing lines in primary cells due to their limitedlife span. The current technology is such that extensivecharacterization is necessary, which eliminates the use of primary cellsbecause of senescence. However, individual Lines of transgenic animalscan be generated by the methods provided herein which produce thepackaging functions, such as gag, pol or env. These lines of animals arethen characterized for expression in either the whole animal or targetedtissue through the selective use of housekeeping or tissue-specificpromoters to transcribe the packaging functions. The vector to bedelivered is also inserted into a line of transgenic animals with atissue-specific or housekeeping promoter. As discussed above, the vectorcan be driven off such a promoter substituting for the U3 region of the5′ LTR (FIG. 3). This transgene could be inducible or ubiquitous in itsexpression. This vector, however, is not packaged. These lines ofanimals are then mated to the gag/pol/env animal and subsequent progenyproduce packaged vector. The progeny, which are essentially identical,are characterized and offer an unlimited source of primary producingcells. Alternatively, primary cells making gag/pol and env and derivedfrom transgenic animals can be infected or transfected in bulk withretrovirus vectors to make a primary cell producer line. Many differenttransgenic animals or insects could produce these vectors, such as mice,rats, chickens, swine, rabbits, cows, sheep, fish and flies. The vectorand packaging genomes would be tailored for species infectionspecificity and tissue-specific expression through the use oftissue-specific promoters and different envelope proteins. An example ofsuch a procedure is illustrated in FIG. 4.

Although the following examples of transgenic production of primarypackaging lines are described only for mice, these procedures can beextended to other species by those skilled in the art. These transgenicanimals may be produced by microinjection or gene transfer techniques.Given the homology to MLV sequences in mice genome, the final preferredanimals would not be mice.

EXAMPLE 4 Production of Gag/Pol Proteins Using Housekeeping Promotersfor Ubiquitous Expression in Transgenic Animals

An example of a well-characterized housekeeping promoter is the HPRTpromoter. HPRT is a purine salvage enzyme which expresses in alltissues. (See Patel et al., Mol. Cell Biol. 6:393–403, 1986 and Meltonet al., Proc. Natl. Acad. Sci. 81:2147–2151, 1984). This promoter isinserted in front of various gag/pol fragments (e.g., Bal I/Sca I; AatII/Sca I; Pst I/Sca I of MOMLV; see RNA Tumor Viruses 2, Cold SpringHarbor Laboratory, 1985) that are cloned in Bluescript plasmids(Strategene, Inc.) using recombinant DNA techniques (see Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982).The resulting plasmids are purified (Maniatis et al., op. cit.) and therelevant genetic information isolated using Geneclean (Bio 101) orelectroelution (see Hogan et al. (eds.), Manipulating the Mouse Embryo:A Laboratory Manual, Cold Spring Harbor, 1986).

These fully characterized DNAs are microinjected in the pronucleus offertilized mouse ova at a concentration of 2 ug/ml. Live-born mice arescreened by tail blot analyses (see Hogan et al., op. cit.).Transgenic-positive animals are characterized for expression levels ofgag/pol proteins by immunoprecipitation of radiolabeled primary cells,such as fibroblast (see Harlow et al. (eds.), Antibodies: A LaboratoryManual, Cold Spring Harbor, 1988). Animals then bred to homozygosity forestablishment of animal lines that produce characterized levels ofgag/pol.

EXAMPLE 5 Production of env Proteins/Hybrid Envelope Proteins UsingHousekeeping Promoters for Ubiquitous Expression in Transgenic Animals

This example utilizes the HPRT promoter for expression of eitherenvelope or hybrid envelope proteins. The envelope proteins can be fromany retrovirus that is capable of complementing the relevant gag/pol, inthis case that of MLV. Examples are ecotropic MLV, amphotrophic MLV,xenotropic MLV, polytropic MLV, or hybrid envelopes. As above, theenvelope gene is cloned behind the HPRT promoter using recombinant DNAtechniques (see Maniatis et al., op. cit.). The resulting “minigene” isisolated (see Hogan et al., op. cit.), and expression of envelopeprotein is determined (Harlow et al., op. cit.). The transgenic envelopeanimals are bred to homozygosity to establish a well-characterizedenvelope animal.

EXAMPLE 6 Production of gag/pol-env Animals Using Housekeeping Promotersfor Ubiquitous Expression in Transgenic Animals

This uses the well-characterized gag/pol animals, as well as the animalsfor the establishment of a permanent gag/pol/envelope animal line. Thisinvolves breeding to homozygosity and the establishment of awell-characterized line. These lines are then used to establish primarymouse embryo lines that can be used for packaging vectors in tissueculture. Furthermore, animals containing the retroviral vector are bredinto this line.

EXAMPLE 7 Production of Tissue-Specific Expression of gag/pol-env orHybrid Envelope in Transgenic Animals

This example illustrates high level expression of the gag/pol, envelope,or hybrid envelope in specific tissues, such as T-cells. This involvesthe use of CD2 sequences (see Lang et al., EMBO J. 7:1675–1682, 1988)that give position and copy number independence. The 1.5 kb Bam HI/HindIII fragment from the CD2 gene is inserted in front of gag/pol,envelope, or hybrid envelope fragments using recombinant DNA techniques.These genes are inserted into fertilized mouse ova by microinjection.Transgenic animals are characterized as before. Expression in T-cells isestablished, and animals are bred to homozygosity to establishwell-characterized lines of transgenic animals. Gag/pol animals aremated to envelope animals to establish gag/pol-env animals expressingonly in T-cells. The T-cells of these animals are then a source forT-cells capable of packaging retroviral vectors. Again, vector animalscan be bred into these gag/pol-env animals to establish T-cellsexpressing the vector.

This technique allows the use of other tissue-specific promoters, suchas milk-specific (whey), pancreatic (insulin or elastase), or neuronal(myelin basic protein) promoters. Through the use of promoters, such asmilk-specific promoters, recombinant retroviruses may be isolateddirectly from the biological fluid of the progeny.

EXAMPLE 8 Production of Either Housekeeping or Tissue-SpecificRetroviral Vectors in Transgenic Animals

The insertion of retroviruses or retroviral vectors into the germ lineof transgenic animals results in little or no expression. This effect,described by Jaenisch (see Jahner et al., Nature 298:623–628, 1982), isattributed to methylation of 5′ retroviral LTR sequences. This techniquewould overcome the methylation effect by substituting either ahousekeeping or tissue-specific promoter to express the retroviralvector/retrovirus. The U3 region of the 5′ LTR, which contains theenhancer elements, is replaced with regulatory sequences fromhousekeeping or tissue-specific promoters (see FIG. 20). The 3′ LTR isfully retained, as it contains sequences necessary for polyadenylationof the viral RNA and integration. As the result of unique properties ofretroviral replication, the U3 region of the 5′ LTR of the integratedprovirus is generated by the U3 region of the 3′ LTR of the infectingvirus. Hence, the 3′ is necessary, while the 5′ U3 is dispensable.Substitution of the 5′ LTR U3 sequences with promoters and insertioninto the germ line of transgenic animals results in lines of animalscapable of producing retroviral vector transcripts. These animals wouldthen be mated to gag/pol-env animals to generate retroviral-producinganimals (see FIG. 4).

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A retroviral packaging cell line, wherein the packaging cell isgenerated from a human host cell line, and wherein the retroviralpackaging cell line provides gag/pol or gag/pol and env.
 2. Theretroviral packaging cell lines of claim 1, that provides gag/pol andenv.
 3. The retroviral packaging cell lines of claim 2, wherein the envis VSV-G.
 4. The retroviral packaging cell lines of claim 1, wherein theretroviral packaging cell line is a non-MLV retroviral packaging cellline.
 5. The retroviral packaging cell lines of claim 4, wherein theretroviral packaging cell line is a retroviral packaging cell lineselected from the group consisting of an RSV packaging cell line, anMMTV packaging cell line, and an HIV packaging cell line.